Adult zebrafish were maintained in a fish auto culture system (Haishen, China) at 28°C, and the light cycle was 14-hours (hrs) on and 10-hrs off. Zebrafish embryos were cultured in 10% Hank’s solution which contained 140 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4 and 4.2 mM NaHCO3 (pH 7.2). AB wild-type and Tg(fli1a:EGFP) transgenic zebrafish were used in this study. The zebrafish handling procedures were approved by Nanjing University School of Medicine.
The human HCC cell lines HepG2, Hep3B, Huh7, and the normal hepatic cell line LO2 were used in this study. All cell lines were obtained from CUNMAI Biotechnologies (Shanghai, China) in 2019 and all cell lines were authenticated by STR test. And all cell lines were tested for mycoplasma contamination by PCR. LO2 cells were cultured in 1640. HepG2, Hep3B, and Huh7 cells were cultured in DMEM. Both media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin and then cultured in a humidified air atmosphere containing 5% carbon dioxide at 37 ℃.
RNA extraction and qRT-PCR
Total RNA was extracted from cell lines using TRIzol reagent. Total RNA was reverse transcribed to cDNA using 1st Strand cDNA Synthesis SuperMix for qPCR kit (Takara). We performed real-time PCR analyses using SYBR Green Master Mix kit (Takara) following the instructions to detect the expression levels of HOXD-AS1 in different liver cell lines. Data were analyzed based on the ∆∆CT method and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control. The sequences for gene-specific primers were HOXD-AS1-F: 5´-ATTCGTCTGACTTGGCTCTT-3´and HOXD-AS1-R: 5´-CCTGTTTTGACCTTTTCCTG-3´. The GAPDH sequences were GAPDH-F: 5´-GGGAGCCAAAAGGGTCAT-3´and GAPDH-R: 5´-GAGTCCTTCCACGATACCAA-3´.
Cell transfection of siRNA and miRNA inhibitor
HepG2, Hep3B and Huh7 cell lines were cultured in six-well plates, then specific siRNAs or miRNA inhibitor were transfected after 24 hrs using Lipofectamine 3000 reagents (Thermo Fisher Scientific, USA). We purchased two HOXD-AS1 siRNAs, miR-130a-3p inhibitor and negative control (NC) siRNA from General Biosystems (China). The HOXD-AS1 siRNA sequences  are as follow: si1-HOXD-AS1: 5’-GAAAGAAGGACCAAAGTAA-3’, si2-HOXD-AS1: 5’-GCACAAAGGAACAAGGAAA-3’, and the NC siRNA sequence are 5’-TTCTCCGAACGTGTCACGT-3’. The sequence of miR-130a-3p inhibitor is AUGCCCUUUUAACAUUGCACUG . Cells were harvested at 24 hrs after transfection, and silencing efficiencies were analyzed by qRT-PCR.
Cell proliferation assays
Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, DOJINDO, Japan). HOXD-AS1 and NC siRNA were transfected into Hep3B and Huh7 cell lines, then cultured in 6-well plates for 24 hrs. A total of 2×103 cells were placed in each well of 96-well plates. Cell proliferation was monitored by measuring the optical density (OD) at 450 nm every 24 hrs according to the manufacturer's instructions, from 0 to 96 hrs, then we analyzed data of each group.
Cell invasion assays
Hep3B and Huh7 were transfected with HOXD-AS1 and NC siRNA. After 24 hrs, transfected cells were plated in 24-well plates with 8-mm-pore size chamber inserts. 2.0×104 or 6.0×104 cells were plated into the upper chambers, which was diluted with the serum-free culture medium. Then the transwell was put into the lower chamber, which including 800 μL medium with 10% FBS. After 24 hrs, the cells migrated to the bottom surface of the membrane, then the cells were fixed with methanol, and stained with 0.1% crystal violet for 30 minutes. The images were taken under the microscope for analysis.
Zebraﬁsh xenograft models
Before injection, cancer cells were labeled with fluorescent dye CM-DiI (Invitrogen, USA). The detailed protocol was as follows: cells were collected, then washed three times with HBSS. The cells were then labeled with CM-DiI at 37 ℃ for 5 min, following by 15 min at 4 ℃. Next, the unincorporated dye was removed by rinsing three times with HBSS and the labeled cells were prepared for injection. The labeled cancer cells were injected into the perivitelline space (PVS) of 48-hpf (hrs post fertilization) zebrafish larvae. Each zebrafish larvae were mounted in 1.2% low-melting gel, and about 300-400 cells were implanted into PVS by the micro-injector. After injection, the zebrafish larva xenografts were cultured at 34℃ until the end of experiments. At 1 day post injection (dpi), the successfully injected xenografts with similar tumor size were selected for the following experiments.
In vivo imaging and quantitative analysis
At 4 dpi, the zebrafish larvae were mounted in 1.2% low-melting gel, and the images were taken by stereotype microscope (MVX10, Olympus, Japan) or confocal microscope using a 20X water-immersion objective lens (Fluoview 3000, Olympus, Japan). The spatial resolution of the images was 1600×1200 (MVX10) or 1024×1024 pixels (Fluoview 3000). The images taken by stereotype microscope were quantitatively analyzed by ImageJ software.
Statistical analysis was performed using unpaired Student’s t-tests. A level of p<0.05 was considered to be statistically significant. Results were displayed as the mean ±SEM.